Periodic Reporting for period 4 - MitoMethylome (Mitochondrial methylation and its role in health and disease)
Période du rapport: 2021-10-01 au 2022-09-30
Methylation is the covalent attachment of a methyl group (CH3) by a specific methyl transferase to biomolecules, such as nucleic acids, proteins, lipids, co-factors, or metabolites. The consequences of such modifications vary from signalling to increased or decreased stability of tertiary structures, and even function. Although much is known about the role of methylation in our cells, within mitochondria its function is less established and was limited to just a handful of known targets. Furthermore, the impact of the mitochondrial methylation potential on cell differentiation and cell function is unknown. The predominant intracellular methyl group donor is S-adenosylmethionine (SAM), which is synthesised in the one-carbon cycle in the cytosol. For methylations inside mitochondria, SAM needs to be imported into mitochondria via a specific transporter. The genetic manipulation of this transporter in model organisms allows me to determine the role of intramitochondrial SAM, its targets, and its physiological consequences.
To aim 1: Develop model systems with intra-mitochondrial SAM deficiency.
My laboratory has generated and characterised genetically modified fruit fly models with varying degrees of intra-mitochondrial SAM deficiency, by replacing the endogenous mitochondrial SAM transporter (SAMC) with functionally compromised versions (Schober et al. 2021a PMID:33608280, Schober et al. 2022 PMID: 35024855). This allowed us to report a differential sensitivity of mitochondrial SAM-dependent reactions to reduced mitochondrial methylation potential.
We have also generated a conditional knockout mouse model that allows for the tissue-specific deletion of murine SAMC. We have generated full body, heart-, and skeletal muscle-specific SAMC knockout mice and are currently preparing for the deletion of SAMC in hepatocytes. Mouse embryonic fibroblasts (MEFs) were isolated from SAMC KO and control embryos and serve as an excellent cell model to investigate the long-term effects of a mitochondrial SAMC deficiency (Schober et al. 2021a PMID:33608280).
To aim 2: Characterise the molecular and metabolic consequences of a depleted intra-mitochondrial SAM pool.
Effects on mitochondrial function, gene, and protein expression, as well as changes in metabolite levels been studied in SAMC KO fly and KO MEF models and published (Schober et al. 2021a PMID:33608280, Schober et al. 2022 PMID: 35024855).
The tissue-specific loss of SAMC in skeletal muscle or heart tissue presents with a surprisingly diverse phenotypic and molecular response. We therefore suggest that the tissue-specific disruption of SAMc is a suitable model to study the mechanism behind the tissue specificity of many diseases (Rumyantseva et al. in preparation, Glasgow et al. in preparation).
Skeletal muscle-specific deletion of SAMC leads to a progressive mitochondrial dysfunction, driven by a continuous loss of mitochondrial gene expression. In combination with studies using SAMC KO MEFs we demonstrate that reduced levels of mitochondrial SAM led to an inefficient mitochondrial gene expression system, exposing intermediate steps of mitochondrial gene expression. Using nanopore sequencing and Cryo-EM technology in combination with mas-spectrometry-based proteomics, we can study previously unknown processing and assembly intermediates (Glasgow et al. in preparation).
To aim 3: Determine the intra-mitochondrial methylated proteome.
We have developed a novel method to identify post-translational modifications, including methylations, in the fly. As a proof-of-principle we described the total phosphoproteome under control and pathological conditions (Schober et al. 2020 PMID: 33230767, Schober et al. 2021b PMID: 33640490). This method was used to determine the mitochondrial methylproteome from flies, with validation of numerous sites in mouse and human samples. (Schober et al. 2021a PMID:33608280).
We identified PNPase as a mitochondrial methylated protein. PNPase is part of the mitochondrial RNA degradation machinery, and we could show that its deletion results in the accumulation and release of double stranded RNAs from mitochondria into the cytosol, which correlates with the induction of an immune response (Pajak et al. 2019 PMID: 31365523).
We identified that the uncharacterised mitochondrial protein ANGEL2 is methylated and could demonstrate that ANGEL2 is required for the dephosphorylation of 3’ termini of three mitochondrial transcripts. This is the first report of such modifications inside mitochondria and our work explains how these transcripts are processed, solving a 40-year-old mystery (Clemente et al. 2022 PMID: 36180430).
To aim 4: Identify modulators of the mitochondrial methylome.
We intended to screen for genetic modulators of the intramitochondrial SAM pool in a genome-wide deficiency screen. The global events of 2020 to 2021 prevented us from committing the necessary daily workload required to perform the planned screen. We therefore readdressed this aim with a novel approach to potentially generate a similar type of information.
Proteomic and transcriptomic data revealed that the cellular one carbon metabolism (1CC) was sensitive to changes in the intramitochondrial SAM pool. We therefore tested whether we could modulate the intramitochondrial SAM pool by providing appropriate intermediates to the fly diet. We could demonstrate that exogenous SAM or methionine supplementation was beneficial for some patient relevant SAMC variants, suggesting a treatment strategy (Schober et al. 2021a PMID:33608280, Schober et al. 2022 PMID: 35024855).
To further test whether mitochondrial dysfunctions are affecting the mitochondrial methylation potential, we performed proteome analysis of primary fibroblasts from patients with mitochondrial disease and controls. The same samples were also investigated for intramitochondrial protein methylation by targeted proteomics. All samples have been run and the data is currently being analysed (Correia et al in preparation). This investigation also included a patient deficient in NDUFB7, a mitochondrial complex I subunit, known to be methylated (Correia et al 2021 PMID: 33502047).
My laboratory has generated and characterised genetically modified fruit fly models with varying degrees of intra-mitochondrial SAM deficiency, by replacing the endogenous mitochondrial SAM transporter (SAMC) with functionally compromised versions (Schober et al. 2021a PMID:33608280, Schober et al. 2022 PMID: 35024855). This allowed us to report a differential sensitivity of mitochondrial SAM-dependent reactions to reduced mitochondrial methylation potential.
We have also generated a conditional knockout mouse model that allows for the tissue-specific deletion of murine SAMC. We have generated full body, heart-, and skeletal muscle-specific SAMC knockout mice and are currently preparing for the deletion of SAMC in hepatocytes. Mouse embryonic fibroblasts (MEFs) were isolated from SAMC KO and control embryos and serve as an excellent cell model to investigate the long-term effects of a mitochondrial SAMC deficiency (Schober et al. 2021a PMID:33608280).
To aim 2: Characterise the molecular and metabolic consequences of a depleted intra-mitochondrial SAM pool.
Effects on mitochondrial function, gene, and protein expression, as well as changes in metabolite levels been studied in SAMC KO fly and KO MEF models and published (Schober et al. 2021a PMID:33608280, Schober et al. 2022 PMID: 35024855).
The tissue-specific loss of SAMC in skeletal muscle or heart tissue presents with a surprisingly diverse phenotypic and molecular response. We therefore suggest that the tissue-specific disruption of SAMc is a suitable model to study the mechanism behind the tissue specificity of many diseases (Rumyantseva et al. in preparation, Glasgow et al. in preparation).
Skeletal muscle-specific deletion of SAMC leads to a progressive mitochondrial dysfunction, driven by a continuous loss of mitochondrial gene expression. In combination with studies using SAMC KO MEFs we demonstrate that reduced levels of mitochondrial SAM led to an inefficient mitochondrial gene expression system, exposing intermediate steps of mitochondrial gene expression. Using nanopore sequencing and Cryo-EM technology in combination with mas-spectrometry-based proteomics, we can study previously unknown processing and assembly intermediates (Glasgow et al. in preparation).
To aim 3: Determine the intra-mitochondrial methylated proteome.
We have developed a novel method to identify post-translational modifications, including methylations, in the fly. As a proof-of-principle we described the total phosphoproteome under control and pathological conditions (Schober et al. 2020 PMID: 33230767, Schober et al. 2021b PMID: 33640490). This method was used to determine the mitochondrial methylproteome from flies, with validation of numerous sites in mouse and human samples. (Schober et al. 2021a PMID:33608280).
We identified PNPase as a mitochondrial methylated protein. PNPase is part of the mitochondrial RNA degradation machinery, and we could show that its deletion results in the accumulation and release of double stranded RNAs from mitochondria into the cytosol, which correlates with the induction of an immune response (Pajak et al. 2019 PMID: 31365523).
We identified that the uncharacterised mitochondrial protein ANGEL2 is methylated and could demonstrate that ANGEL2 is required for the dephosphorylation of 3’ termini of three mitochondrial transcripts. This is the first report of such modifications inside mitochondria and our work explains how these transcripts are processed, solving a 40-year-old mystery (Clemente et al. 2022 PMID: 36180430).
To aim 4: Identify modulators of the mitochondrial methylome.
We intended to screen for genetic modulators of the intramitochondrial SAM pool in a genome-wide deficiency screen. The global events of 2020 to 2021 prevented us from committing the necessary daily workload required to perform the planned screen. We therefore readdressed this aim with a novel approach to potentially generate a similar type of information.
Proteomic and transcriptomic data revealed that the cellular one carbon metabolism (1CC) was sensitive to changes in the intramitochondrial SAM pool. We therefore tested whether we could modulate the intramitochondrial SAM pool by providing appropriate intermediates to the fly diet. We could demonstrate that exogenous SAM or methionine supplementation was beneficial for some patient relevant SAMC variants, suggesting a treatment strategy (Schober et al. 2021a PMID:33608280, Schober et al. 2022 PMID: 35024855).
To further test whether mitochondrial dysfunctions are affecting the mitochondrial methylation potential, we performed proteome analysis of primary fibroblasts from patients with mitochondrial disease and controls. The same samples were also investigated for intramitochondrial protein methylation by targeted proteomics. All samples have been run and the data is currently being analysed (Correia et al in preparation). This investigation also included a patient deficient in NDUFB7, a mitochondrial complex I subunit, known to be methylated (Correia et al 2021 PMID: 33502047).
My laboratory has developed several genetically modified fly models to study intramitochondrial SAM, including null, knock-in, and reporter flies. All fly lines have been deposited at a central Drosophila depository (Bloomington research centre). We are continuing to generate model fly lines for methylated mitochondrial proteins, expressing mimetics as well as tagged versions. All lines will become publicly available once published.
We have established genetically modified mouse models, and generated tissue-specific KO mouse models, as well as a complete body KO. None of these models have existed prior to this project.
My group developed and improved a stable-isotope labelling technique to be used in fruit fly models, which is applicable for any researcher interested in in vivo labelling or tracing. The technique vastly improves efficiency and sensitivity of currently available methods and is applicable for the identification of a range of different protein modifications or other labels (Schober et al. 2020 PMID: 33230767, Schober et al. 2021b PMID: 33640490).
Our work suggests that a substantial portion of the mitochondrial methylproteome is methylated already prior to mitochondrial import. This proposes a new regulatory mechanism for mitochondrial function.
We have established genetically modified mouse models, and generated tissue-specific KO mouse models, as well as a complete body KO. None of these models have existed prior to this project.
My group developed and improved a stable-isotope labelling technique to be used in fruit fly models, which is applicable for any researcher interested in in vivo labelling or tracing. The technique vastly improves efficiency and sensitivity of currently available methods and is applicable for the identification of a range of different protein modifications or other labels (Schober et al. 2020 PMID: 33230767, Schober et al. 2021b PMID: 33640490).
Our work suggests that a substantial portion of the mitochondrial methylproteome is methylated already prior to mitochondrial import. This proposes a new regulatory mechanism for mitochondrial function.